The human eye detects color through cone photoreceptor cells in the retina. Human color vision is enabled by three types of cone cells defined by expression of light-detecting opsin proteins (L-opsin/red, M- opsin/green, and S-opsin/blue) arranged in a random pattern in the retina. Trichromatic color vision including red, green, and blue cone cells in the retina is exclusive to humans, apes, and Old World monkeys. Mutations in cone cell development and maintenance cause color blindness, retinitis pigmentosa, cone dystrophy, and macular degeneration. The goal of this project is to determine the mechanisms controlling the specification of cone subtype fates in the human eye using a powerful system that we have adapted to differentiate human retinal organoids from stem cells. These ?retinas in a dish? recapitulate human photoreceptor development in gene expression, developmental timing, and morphology. Previous data suggests that the generation of red, green, and blue cone cells occurs through a two-step decision process. First, there is a decision to choose either the blue or red/green fate, and then a second decision to become either a red or green cone cell. The decision to be either a red or green cone cell occurs at the gene locus level on the X chromosome, where a locus control region (LCR) enhancer element lies upstream of the red opsin and green opsin genes. We have analyzed RNA sequencing data from human fetal samples and human stem cell-derived retinal organoids and showed that green opsin expression occurs first in development. We hypothesize that: (1) the decision between green and red cones is temporal during human development, with green cone cells generated before red cone cells (Aims 1 and 2), (2) a map of cone cell distributions in the human retina will reveal more green cones at the early born central retina and more red cones at the late born periphery (Aim 1), and that timing of retinoic acid and levels of thyroid hormone signaling are responsible for the generation of red cone cells (Aim 2). We will label green and red cone cells in human and fetal eyes using an RNA in situ hybridization technique that successfully distinguishes green and red opsin expression, and use computational modeling to analyze a map of the whole retina (Aim 1). We will determine how retinoic acid and thyroid hormone affects specification of green and red cones by modulating the timing of retinoic acid and the levels of thyroid hormone in organoids, and test a mechanism by which these signals affect the generation of green and red cone cells (Aim 2). This project will be carried out at Johns Hopkins University in the Department of Biology in the lab of Robert J. Johnston Jr. The applicant will receive additional training from collaborators at Johns Hopkins University (Dr. James Taylor, Dr. Elijah Roberts) and Medical School (Dr. Don Zack). This project will elucidate how the temporal mechanisms behind cone cell subtype fate choice are regulated during human retinal development, contributing to our general understanding of gene regulatory and disease mechanisms.